Preparation of a Compound Dilution Series

Use of Digital Dispensing to Facilitate an Accurate, Fast, and Facile Workflow

The pipetting of serial dilution series is daily business in biomedical laboratories, be it for the determination of the effect of test compounds in biochemical or cellular assays, for finding optimal concentrations of substrates and co-factors in assay development, or for a wide range of enzymological studies, such as active site titrations or determinations of kinetic constants of slow binding and irreversible inhibitors.

In this article, we introduce a benchtop instrument, HP D300 Digital Dispenser, available from Tecan, that reliably and rapidly dispenses small volumes of DMSO-dissolved compounds directly into wells of microtiter plates in a contact-free manner directly from stock solutions. The device dispenses single droplets of either 20 or 13 picoliters, and larger volumes are digitally built by rapidly shooting multiple droplets into the same well, just like ink-jet printers shoot ink droplets on paper to print letters and words.

The remarkably small volume that is transferred with a single shot allows the direct dispensing of stock solutions into assay plates, spanning a wide range of concentrations of the dispensed compound. It is possible to generate custom-made dilution series of DMSO-dissolved compounds rapidly and accurately, with minimized compound consumption and with minimal effort.

Operating the instrument is simple. First, parameters such as the plate format (12- to 384-well plates are supported), the assay volume, the maximally tolerated DMSO concentration, and the names and stock concentrations of the compounds to be dispensed are defined. The simplest way to subsequently program a dilution series is to highlight a certain number of wells, and then define the desired starting and end concentrations of the compound and decide whether the dilution scheme should be logarithmic or linear.

When starting the dispensing procedure, the program asks the user to pipet the compound into a dispensehead reservoir (usually a few microliters), and the instrument then shoots the compound directly into the wells of the microtiter plate as programmed. Completely custom-made dilution series or complicated matrix experiments can thus be prepared in microtiter plates in a few minutes with minimal manual work.

In this tutorial, we show two different applications for the use of digital dispensing, namely the determination of kinetic parameters of enzyme-catalyzed reactions and the direct dispensing of test compounds from a DMSO stock solution onto living cells.

Materials, Methods, and Results

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Figure 1. (A) Four different volumes of a 2 mM fluorescein stock solution were dispensed with all eight dispenseheads of a cassette into wells prefilled with 25 µL phosphate-buffered saline (PBS) with 10 repeats. The arrow indicates the dispensing pattern. (B) The eight individual plots show the measured fluorescence intensities for each well for each of the eight blocks pipetted by the eight dispenseheads of a cassette. The fluorescein signal was recorded with a microtiter plate reader with an excitation wavelength of 495 nm, and an emission wavelength of 520 nm. (C) Graph depicting the dispensed four volumes of fluorescein against the average of all 80 measured fluorescence intensity values per volume. The volume correlates nicely with the fluorescence intensities, as can be seen from linear regression, and the standard deviations are small (6.5, 2.4, 4.3, and 7.2% for 25, 4, 0.6, and 0.1 nL, respectively).

In order to test the accuracy and reproducibility of the instrument, we performed a simple experiment in which multiple copies of four different concentrations of fluorescein were dispensed into a 384-well plate with all eight dispenseheads of a cassette. Each concentration was dispensed in 10 repeats, and the fluorescence intensities were measured for each well (Figure 1A).

The reproducibility and accuracy of digital dispensing was very high (Figure 1B and 1C); however, it is worth noting that the seemingly biggest error stems from the very first well with which the dispensing starts (Figure 1A and 1B). Knowing this, one can optionally start dispensing into a “waste” well that is not used in the experiment.

Determination of Kinetic Parameters of Enzyme-Catalyzed Reactions

The standard procedure for the determination of the kinetic constants of enzyme-catalyzed reactions is to mix the enzyme of interest, which is kept at a constant concentration, with varying starting concentrations of the substrate(s) of interest. Progression curves of product formation over time are then recorded, and the initial reaction velocities are plotted against the substrate concentrations. Fitting to the Michaelis-Menten velocity equation yields the kinetic constants kcat (catalytic rate constant) and KM (Michaelis constant).

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Figure 2. (A) The graph depicts initial reaction velocities of a protease-catalyzed reaction at different concentrations of a peptidic substrate. Under steady-state conditions this plot can be fitted with the Michaelis-Menten velocity equation. (vinitial: initial reaction velocity, kcat: catalytic rate constant, [E]: enzyme concentration in the reaction mixture, KM: Michaelis constant, [S]: initial substrate concentration). The fit will produce values for kcat and KM. The ratio kcat / KM, typically referred to as the “specicity constant” or as the “catalytic efficiency ratio,” is a measure of the efficacy with which a given substrate is turned over to product by the enzyme of interest. (See the Michaelis-Menten velocity equation below.) (B) Table of substrate concentrations in the 32 wells of a 384-well plate used for substrate titration in the described experiment (2 significant digits shown).

In the example shown in Figure 2, we have determined the kcat and KM values of a protease-catalyzed hydrolysis of a fluorescently labeled peptide in a homogenous assay system. The advantage of digital dispensing for the determination of kinetic constants is the speed and simplicity with which dilution series of substrates can be dispensed, the low consumption of substrate, and the possibility to set many measurement points in a predefined concentration range with little effort. The simplicity of the procedure allows exhaustive covering of the concentration range of interest.

In the example shown in Figure 2A, we programmed a logarithmic dilution series over two columns of a 384-well plate (i.e., over 32 wells) using a 5 mM compound stock concentration and an assay volume of 25 µL with a starting substrate concentration of 100 µM and an end concentration of 5 nM.

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Michaelis-Menten velocity equation

The resulting serial dilution series has a dilution factor of approximately 1.38 with the three highest substrate concentrations 100, 73, and 53 µM and three lowest substrate concentrations of 10, 7.8, and 5.2 nM (Figure 2B). Due to the nature of digital dispensing, the end concentrations result from discrete dispensing steps of single droplets of the stock solution that have a discrete volume. Therefore, the end concentrations can slightly differ from the programmed ones (as in this example, the lowest concentration is 5.2 instead of 5 nM). However, the real end concentrations are recorded in a report sheet and thus can be taken into account in data analysis.

Direct Dispensing of DMSO Solutions onto Living Cells

The small droplet volume allows the direct dispensing of DMSO solutions onto live cells, circumventing the need of an intermediate dilution step of the DMSO-dissolved test compound in aqueous buffer or cell medium before compound addition onto the cells. Cell viability and fitness is not affected by direct dispensing, while bulk pipetting of DMSO onto living cells has a negative effect on both.

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Figure 3. (A) The graph depicts the effect of an inhibitor in a cellular assay. The readout of the assay is a high-content readout, where with uninhibited cells a fluorescently labeled protein translocates into the nucleus (B, top panel), and in the fully inhibited cells, the fluorescently labeled protein is located in the cytoplasm (B, bottom panel). The recorded IC50 for the selected compound is slightly better when using digital dispensing compared to an intermediate dilution step, indicating that there may be a slight loss of compound during the extra steps of dilution series preparation and intermediate dilution. For the bulk of the inhibitors we have tested, no significant IC50 difference was observed (data not shown).

We compared the direct dispensing of DMSO-dissolved inhibitors onto cells with a standard process in which a primary serial dilution series (in DMSO) was transferred first into an intermediate dilution plate (aqueous medium) before pipetting the inhibitor onto cells. The resulting compound response curves are similar (Figure 3).

Circumventing an intermediate dilution step avoids the risk of precipitation of the test compound during the intermediate dilution step, which can lead to compound loss. Additionally, direct dispensing allows the facile addition of any kind of DMSO-dissolved compounds onto cells, like, for example, an inducer, without significantly affecting the volume of the culture media in the wells.

Conclusion

We routinely use the HP D300 Digital Dispenser for the rapid generation of customized titrations, e.g., of substrates for KM determinations or of inhibitors for IC50 determinations, or for active site titrations in the context of assay development for in vitro biochemical assays. The small droplet volume allows the direct dispensing of DMSO solutions onto live cells without compromising cell viability due to the dispensing process. The device is robust with a small footprint as a standalone device, and the control software is intuitive and easy to use.

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